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Despite its low water solubility, esculetin (EC) have been described to demonstrate various health benefits. Thus, we sought to develop esculetin-loaded mixed micelles (EC-M) delivery system to purposively improve biological availability and anti-hyperglycemia activity of EC. Thin-film hydration method was employed to fabricate EC-M, amid characterization with transmission electron microscopic analysis (TEM), coupled with physical properties such as particle size (PS), poly-dispersity index (PDI), zeta-potential (ZP) and stability testing. We analyzed in-vitro release and studied EC-M pharmacokinetics in rats. The hyperglycemic mice model was established with streptozotocin (STZ) to evaluate anti-hyperglycemic activity of EC-M. The PS, PDI and ZP of EC-M were 47.97 ± 0.41 nm, 0.189 ± 0.005 and -25.55 ± 0.28 mV, respectively. The release rate of EC-M increased comparable to free EC in the three media. The oral biological availability and half-life of EC-M increased respectively by 3.06 and 1.45 folds compared to free EC. Besides, we observed 46.21% decrease in blood glucose of mice in EC-M group comparable to the model control, wherein, the anti-hyperglycemic effect of EC-M was better compared to free EC. Conclusively, EC-M may ideally serve as a novel approach to enhance biological availability and increased anti-hyperglycemic activity of EC. [ABSTRACT FROM AUTHOR]

A cyclometalated ruthenium (II) complex 1 [(Ru (Phen)2(Pbznh)]+ PF6− (Phen = 1,10‐phenanthroline and Pbznh = N‐(4‐(pyridine‐2‐yl)benzylidene) nicotinohydrazide) with nicotinohydrazide as a functional group was synthesized and characterized. Changes of its absorption spectra and color induced by Cu2+ and Fe3+ were systematic investigated. The results demonstrated that complex 1 could be served as a colorimetric probe to fast, selective and sensitive detection of Cu2+ and Fe3+ both in acetonitrile and filter paper based strips. Upon addition of Cu2+ and Fe3+ to solution of probe 1, solution color changed from pink to colorless and light yellow respectively, and their corresponding detection limit were calculated to be 3.26 × 10−8 M and 3.12 × 10−7 M. Moreover, color of test papers with 1 changed from pink to colorless/yellow when Cu2+/Fe3+ were dropwise added. Therefore, it can be used as a desirable 'naked‐eye' indicator candidate for Cu2+ and Fe3+. [ABSTRACT FROM AUTHOR]

Aerosol optical depth (AOD) is the most basic parameter that describes the optical properties of atmospheric aerosols, and it can be used to indicate aerosol content. In this study, we assimilated AOD data from the Fengyun-3A (FY-3A) and MODIS meteorological satellite using the Gridpoint Statistical Interpolation three-dimensional variational data assimilation system. Experiments were conducted for a dust storm over East Asia in April 2011. Each 0600 UTC analysis initialized a 24-hWeather Research and Forecasting with Chemistry model forecast. The results generally showed that the assimilation of satellite AOD observational data can significantly improve model aerosol mass prediction skills. The AOD distribution of the analysis field was closer to the observations of the satellite after assimilation of satellite AOD data. In addition, the analysis resulting from the experiment assimilating both FY-3A/MERSI (Medium-resolution Spectral Imager) AOD data and MODIS AOD data had closer agreement with the ground-based values than the individual assimilation of the two datasets for the dust storm over East Asia. These results suggest that the Chinese FY-3A satellite aerosol products can be effectively applied to numerical models and dust weather analysis. [ABSTRACT FROM AUTHOR]

We report a strategy for the self-assembly of zeolitic imidazolate framework-8 (ZIF-8) particles induced by the evaporation of a methanol–water mixture. This strategy effectively suppresses the coffee ring effect, facilitates the rapid assembly of ZIF-8 particles, and improves the orientation and optical properties of self-assembled superstructures. [ABSTRACT FROM AUTHOR]

Introduction: Chronic obstructive pulmonary disease (COPD) is a common, irreversible but preventable disease characterized by persistent respiratory symptoms. The mortality rate of COPD is predicted to reach 5.4 million by the year 2060. Despite its heavy burden on healthcare expenditure worldwide, only 15% of cases are medically identified. The potential benefits of facemask-wearing for COPD patients remain a topic of debate. Methods: We will conduct a systematic review of all randomized trials and non-randomized controlled trials to evaluate the impact of facemasks on COPD patients. Our review will be based on literature obtained through a comprehensive search strategy across multiple electronic databases, including the Cochrane Library, Embase, PubMed, Web of Science, the Chinese Biomedical Database (SinoMed), and China National Knowledge Infrastructure (CNKI), with no restrictions on language or date of publication. Two independent researchers will extract and assess all relevant data using pre-designed data extraction forms. The included studies will be assessed using the Cochrane RoB2 tool and the suggested risk of bias criteria proposed by the Effective Practice and Organization of Care reviews group of the Cochrane collaboration. The quality of evidence will be assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. We will use Review Manager 5.4 software for statistical analysis. Discussion: In the context of COVID-19, it is important for COPD patients to wear facemasks. This study aims to conduct a comprehensive and systematic assessment of the impact of facemasks on the physiology and activity of COPD patients. Trial registration: PROSPERO registration numberCRD42022326265. [ABSTRACT FROM AUTHOR]

The structures and energies of complexes obtained upon interaction between glutathione (GSH) and alkali (Li, Na, K), or alkaline earth metal (Be, Mg, Ca), or group IIIA (Al) cations were studied using quantum chemical density functional theory. The characteristics of the interactions between GSH and the metal cations at different nucleophilic sites of GSH were examined selecting systematically, both mono- and multi-coordinating were taken into account. The results indicated that the heteroatom of GSH, the radius and charge of metal ion, and the coordination number of the metal cation with the ligand played important roles in determining the stability of these complexes. Moreover, the intramolecular hydrogen migration in GSH could be promoted by the metal cations during coordination reaction. Furthermore, the Al cation might catalyze the decarboxylation reaction and stimulate the formation of covalent bond between S atom and adjacent O atom of GSH. [ABSTRACT FROM AUTHOR]

Sepsis is a severe systemic inflammatory response induced by infection. Innate immunity recognizes pathogen components such as lipopolysaccharides (LPS), and mediates the polarization of immune cells and the release of cytokines. However, this process is also crucial for triggering sepsis and septic shock. To investigate the potential therapeutic function of 11H-indeno [1,2–b] quinoxalin-11-one oxime (IQ-1S) to sepsis, LPS plus d -galactosamine was used to establish a sepsis mouse model. Flow cytometry was performed to catalyze T cells and macrophages in mouse spleen. ELISA assay and qRT-PCR assay were performed to estimate the expression levels of cytokines and related genes including TNF-α, IL-6, IL-1β, Nos2, Arg and Mrc. The protein levels of NF-κB, AP1, NF-Y, p-JNK2, JNK2, p-p38, p38, p-IκBα, IκBα, p-IKKβ and IKKβ were evaluated by Western blot assay. IQ-1S treatment significantly reduced mortality and lung inflammation in sepsis mice. IQ-1S treatment decreased the levels of inflammatory cytokines in sepsis mice. Polarization of M1 macrophages was suppressed by IQ-1S in vitro. IQ-1S significantly inhibited the activation of the JNK signaling pathway and reduced the phosphorylation level of JNK2 in sepsis mice. IQ-1S protected the mice against LPS-induced sepsis through inhibiting JNK signaling pathway. [ABSTRACT FROM AUTHOR]

The Fengyun-4A (FY-4A) meteorological satellite, a next-generation geostationary meteorological satellite, was launched on December 11, 2016. For instance, the Advanced Geosynchronous Radiation Imager (AGRI) aboard FY-4A (AGRI/FY-4A) takes full-disk images at a 15-min interval in 14 spectral bands with the 0.5–4-km resolution. Here we developed data assimilation system based on the Gridpoint Statistical Interpolation (GSI) system in which the Aerosol Optical Depth (AOD) derived from FY-4A data were successfully assimilated for the first time. The capability to assimilate FY-4A Aerosol optical depth (AOD) with an hourly cycling configuration was then evaluated by a dust storm over East Asia during 12–14 May 2019. The analyses initialized Weather Research and Forecasting-Chemistry (WRF-Chem) model forecasts. The system is tested with FY-4 AOD, Himawari-8 AOD in experiments and then the results are compared to the Aerosol Robotic Network (AERONET) AOD observations, which serving as the independent observations. The results indicated that assimilating FY-4 AOD substantially showed much better agreement with observations than those from the control. Furthermore, the Bias and RMSE generally reduced about 20% with forecast range. This study indicates that the aerosol data assimilation using data from FY-4A can be used to improve the performance of forecast model. • It is the first attempt to assimilate FY-4A AOD with a rapid-update DA system. • It was investigated for the dust storm occurred over East Asia on 12–14 May 2019. • General positive impacts were achieved from assimilating high-frequency data. [ABSTRACT FROM AUTHOR]

Himawari-8, a next-generation geostationary meteorological satellite, is equipped with the Advanced Himawari Imager (AHI) that provides full-disk images of Earth every 10 min for 16 observation bands from visible to infrared. In this study, the capability to assimilate AHI Aerosol optical depth (AOD) has been developed within the Gridpoint Statistical Interpolation (GSI) system with an hourly cycling configuration and application to a dust storm over East Asia during 5–7 May 2017. Analyses were produced hourly, and 24 h forecasts were produced every 6 h within the Weather Research and Forecasting with Chemistry model. It was found that the mean bias and root-mean-square error (RMSE) after data assimilation is obviously reduced (by about 30%) when compared to the control experiment which didn't assimilate any observations during the dust storm period, which verified the positive effects of AOD data assimilation systems. In addition, aerosol analyses and forecasts with AOD data assimilation were substantially improved when compared to independent AOD observations from AERONET sites. Therefore, the AOD data assimilation system developed here could be used as a tool to generate better dust storm forecasts. • It is the first attempt to assimilate AHI AOD with a rapid-update DA system. • It was investigated for the dust storm occurred over East Asia on 5–7 May 2017. • General positive impacts were achieved from assimilating high-frequency data. [ABSTRACT FROM AUTHOR]

Encapsulation of esculetin into DSPE-MPEG2000 carrier was performed to improve its water solubility and oral bioavailability, as well as enhance its anti-inflammatory effect on a mouse model of ulcerative colitis that was induced with dextran sulphate sodium (DSS). We determined the in-vitro and in-vivo high-performance liquid chromatographic (HPLC) analysis method of esculetin; Esculetin-loaded nanostructure lipid carrier (Esc-NLC) was prepared using a thin-film dispersion method, wherein a particle size analyser was used to measure the particle size (PS) and zeta potential (ZP) of the Esc-NLC, while a transmission electron microscope (TEM) was employed to observe its morphology. Also, HPLC was used to measure its drug loading (DL), encapsulation efficiency (EE) and the in-vitro release of the preparation, as well as investigate the pharmacokinetic parameters. In addition, its anti-colitis effect was evaluated via histopathological examination of HE-stained sections and detection of the concentrations of tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (β), and IL-6 in serum with ELISA kits. The PS of Esc-NLC was 102.29 ± 0.63 nm with relative standard deviation (RSD) of 1.08% (with poly-dispersity index-PDI of 0.197 ± 0.023), while the ZP was −15.67 ± 1.39 mV with RSD of 1.24%. Solubility of esculetin was improved coupled with prolonged release time. Its pharmacokinetic parameters were compared with that of free esculetin, wherein the maximum concentration of the drug in plasma was increased by 5.5 times. Of note, bioavailability of the drug was increased by 1.7 times, while the half-life was prolonged by 2.4 times. In the anti-colitis efficacy experiment, the mice in Esc and Esc-NLC groups exhibited significantly reduced levels of TNF-α, IL-1β, and IL-6 in their sera comparable to the DSS group. Colon histopathological examination revealed that mice with ulcerative colitis in both Esc and Esc-NLC groups displayed improved inflammation, amid the Esc-NLC groups having the best prophylactic treatment effect. Esc-NLC could ameliorate DSS-induced ulcerative colitis by improving bioavailability, prolonging drug release time and regulating cytokine release. This observation confirmed the potential of Esc-NLC to reduce inflammation in ulcerative colitis, albeit the need for follow-up research to verify the application of this strategy to clinical treatment of ulcerative colitis. [ABSTRACT FROM AUTHOR]

Aim: Hydrophobic pinocembrin (PCB) was incorporated into a new nano-drug delivery system to enhance solubility, bioavailability and anti-hyperuricemic activity of the drug. Methods: We fabricated PCB loaded polymeric micelles (PCB-FPM) by thin film dispersion method and appropriately determined their physical characteristics. The oral relative bioavailability and anti-hyperuricemic activity of PCB-FPM and free PCB were observed. Results: The optimum particle size of the micelles was 19.90 ± 0.93 nm. PCB-FPM exhibited great stability within 18 days, coupled with lower cytotoxicity and higher biocompatibility. Moreover, the percent cumulative release of PCB-FPM was much higher than free PCB in the dissolution media. The oral bioavailability of PCB-FPM was increased by 2.61 times compared with free PCB. Uric acid (UA) level of rats was reduced in PCB-FPM group (200 mg/kg) by 78.82% comparable to the model control. Conclusion: PCB-FPM may become an ideal strategy to increase oral in-vivo availability and anti-hyperuricemic activity of PCB. [ABSTRACT FROM AUTHOR]

In recent years, the Chinese mitten crab (Eriocheir sinensis), which is widely farmed in China, has been frequently affected by Hepatospora eriocheir. The gut response of mitten crabs to H. eriocheir infection is not yet known. The aim of this study was to evaluate the effects of H. eriocheir infection on the gut microbiota and proteome of E. sinensis. In this study, E. sinensis was divided into two groups based on whether it was infected with H. eriocheir using PCR and Calcofluor white staining. Calcofluor white staining revealed that H. eriocheir was massively aggregated in the gut. Proteomic results showed that 3959 proteins were identified. Under a 1.2-fold change threshold, 420 differentially expressed proteins (DEPs) were screened, including 292 upregulated DEPs and 128 downregulated DEPs. Differential protein expression related to apoptosis and energy metabolism increased, while protein expression related to immune and inflammatory responses and cell motility decreased. In addition, the microbiota results showed that alpha diversity tended to increase, and beta diversity indicated significant differences in gut microbiota between the two groups. Bacteroidetes, Proteobacteria, and Firmicutes were the dominant phyla in the E. sinensis gut. Compared with the control group, Bacteroidetes were significantly reduced in the infected group, while Firmicutes and Tenericutes were significantly increased. At the genus level, the abundance of Bacteroides , Flavobacterium , Shewanella and Aeromonas decreased significantly, while Candidatus Bacilloplasma , Dysgonomonas , Tyzzerella_3 and Lactovum increased significantly. Microbial function prediction found that most differentially abundant bacteria were related to metabolism. At the same time, correlation analysis between the proteome and gut microbiota found potential roles of differential bacteria and proteins involved in metabolism and immunity. These findings suggest that H. eriocheir infection can alter the gut microbiota composition and proteome of E. sinensis. • Hepatospora eriocheir was massively aggregated in the Eriocheir sinensis gut. • 420 differential proteins were to H. eriocheir infection by TMT proteomics analysis. • Bacteroidetes reduced, while Firmicutes and Tenericutes increased in gut after H. eriocheir infection. • The differential bacteria and protein in H. eriocheir infection were involved in metabolism and immunity. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies on the underlying mechanism for porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive failure have been focused on the viral replication in the endothelial macrophages, and the susceptibility of porcine endometrial endothelial (PEE) cells to PRRSV has not yet been investigated. Therefore, in the present study we generated a PEE cell line by transfection of the primary cells with a SV40 large T antigen expression vector. The PEE cell line maintained the endothelial morphology with a significantly faster growth rate, shorter population doubling time and higher plating efficiency than the primary cells. The endothelial origination of the cell line was confirmed by detection of the endothelial cell-specific markers. The PEE cell line had been passed successively for 60 generations with an unlimited growth potential. To further characterize the PEE cell line, cells of different passages were infected with different PRRSV strains and analyzed for the viral antigen and replication. Overt cytopathic effect was observed from 36h postinfection (HPI) and the viral antigen detected as early as 12 HPI. The infectious virus was recovered from the infected PEE cells with a titer higher than that in MARC-145 cells. Since the data presented indicate a high susceptibility of PEE cells to PRRSV, we conclude that the PEE cell line generated will be useful for growth of PRRSV and further studies on the underlying mechanism for PRRSV infection of PEE cells. The finding of the susceptibility of PEE cells to PRRSV may provide an alternative explanation for PRRSV-induced reproductive failure. [Copyright &y& Elsevier]

African swine fever (ASF), caused by African swine fever virus (ASFV), was first reported in Kenya in 1921, but an effective vaccine or antiviral drug is still not available for ASFV control. Rapid and effective diagnostics are key steps in managing ASF. We generated two monoclonal antibodies (MAbs) against the ASFV phosphoprotein P30 and designated these as 3H7A7 and 6H9A10. Epitope mapping revealed that MAb 3H7A7 and 6H9A10 recognized aa 144-154 and aa 12-18 of P30, respectively. A signal-amplified sandwich colloidal gold test strip for rapid detection of ASFV was developed based using these MAbs. Sensitivity and specificity analysis showed that the detection limit of the strip was 2.16 ng of P30. The strip only reacted with ASFV and did not react with other common porcine viruses. In detection tests using 153 clinical field samples including sera, plasma, anticoagulant-treated blood, and tissue, the strip had 95.42% concordance with real-time PCR. The new MAbs specific for P30 and the rapid colloidal gold test strip helped to reveal novel B cell epitopes in P30 and provide an efficient diagnostic test for on-site clinical detection of ASF. [ABSTRACT FROM AUTHOR]

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris , the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis , the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system. • A novel serine proteases contain two ShK-domains from Eriocheir sinensis , EsShK-SP, was reported first time so far. • The expression of EsShK-SP was significantly up-regulated after Spiroplasma eriocheiris infection. • The over-expression of EsShK-SP increased the proPO transcription and PO activity to resist S. eriocheiris infection. • EsShK-SP RNAi decreased the proPO transcription and PO activity to promote S. eriocheiris infection. [ABSTRACT FROM AUTHOR]

Background: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). Methods: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. Results: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. Conclusion: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine. [ABSTRACT FROM AUTHOR]

• We compare ELP-VLP vaccine efficacy with two commercial PCV2 vaccines. • We demonstrate that ELP-VLP induces stronger VN antibody and IFN-γ responses. • We show that ELP-VLP is more effective against PCV2 infection. Porcine circovirus type 2 (PCV2) is an economically important swine pathogen and vaccination is the primary tool for the disease control. Previously, we developed a more cost-effective PCV2 virus-like particle (VLP) vaccine by using ELPylation technology. In the present study, we compared the ELPylated VLP (ELP-VLP) PCV2 vaccine efficacy with commercial inactivated Yuanlijia vaccine and VLP-based Circoflex vaccine by experimental challenge. After one dose of vaccination with the three different vaccines, ELP-VLP vaccine group showed significantly (p < 0.05) stronger virus neutralizing antibody and interferon-γ responses than the two commercial vaccine groups. All vaccinated pigs showed significant (p < 0.05) improvement in average daily weight gain (ADWG) before challenge. After challenge with PCV2, however, only ELP-VLP-vaccinated pigs showed significant (p < 0.05) improvement in ADWG. All vaccinated pigs showed significant (p < 0.05) reductions in PCV2 loads in the blood, nasal secretion and lymph nodes, ELP-VLP-vaccinated pigs in particular. In addition, vaccination with ELP-VLP vaccine provided stronger protection against pulmonary and lymphoid pathologies than that with the two commercial vaccines. Therefore, ELP-VLP vaccine is more effective to control PCV2 infection than the two commercial vaccines based on clinical, immunological, virological and pathological evaluations. [ABSTRACT FROM AUTHOR]

Abstract In consideration of the nucleophilic capacity of thiophenols, which could cleave sulfonamide bond under neutral conditions, a cyclometalated iridium(III) complex (probe 1) comprised 2,4-dinitrobenzenesulfonamide (DNBS) as the recognition unit was designed and synthesized to high selectivity and sensitivity detect thiophenols successfully based on internal charge transfer (ICT) mechanism. Upon addition with thiophenols to 20% DMSO/PBS solution of nearly non-fluorescent probe 1 (10 μM, pH = 7.4), fluorescent product (complex 2) was formed after cleavage of DNBS group and a significant emission enhancement at 470 nm was observed. In addition, it could be successfully applied to detect thiophenol in water samples with a high recovery, confirming its practical value in environment science. Graphical abstract A cyclometalated iridium (III) complex comprised 2,4-dinitrobenzenesulfonamide (DNBS) as the recognition unit (probe 1) was synthesized and its fluorescent behaviors toward thiophenols in 20% DMSO/PBS solution and water samples were investigated systematically. The results demonstrated that probe 1 could serve as a sensitive and selective fluorescent probe to detect thiophenols and could not be disturbed by aliphatic thiols. Image 1 Highlights • A iridium (Ⅲ) complex probe comprised 2,4-dinitrobenzenesulfonamide as recognition unit was designed and synthesized. • Its detection behaviors toward thiophenols in 20% DMSO/PBS and water samples were investigated systematically. • The mechanisms of recognition of thiophenols had been discussed through 1H NMR spectrum. [ABSTRACT FROM AUTHOR]

Abstract Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability. Highlights • We develop a novel strategy for half-life extension of PoIFN-α by fusing to SPG domain C2. • We optimize the conditions for soluble ELP-PoIFNα-C2 protein expression and purification. • We cleave the ELP tag with active TEV protease aggregate. • We demonstrate significantly improved half-life of PoIFNα-C2 protein in rats. [ABSTRACT FROM AUTHOR]

Highlights • We expressed three ELP-tagged and two His-tagged PoIFNs in E. col. • We optimized conditions for soluble expression and purification of ELP-tagged PoIFNs. • We compared two ELP fusion orders on PoIFN expression and bioactivity. • We demonstrated the solubility of ELP-fused of PoIFNs under the physical conditions. • We demonstrated that ELP-fusion is a feasible strategy for easy purification and half-life extension of rPoIFNs. Abstract The clinical use of recombinant interferons (rIFNs) is limited by higher purification cost and quick clearance from circulation. Elastin-like polypeptides (ELPs) are a novel tag for recombinant protein purification and half-life extension. In this study, we evaluated the feasibility of ELP fusion for simple purification and half-life extension of recombinant porcine IFNs (rPoIFNs). After construction of five different fusion expression vectors, we optimized the conditions for soluble protein expression and purification. SDS-PAGE analysis showed that, unlike PoIFNα-His and PoIFNγ-His, PoIFNα-ELP, ELP-PoIFNα and PoIFNαγ-ELP were expressed mainly as soluble proteins at 20 ℃. The optimal conditions for the inverse transition cycling (ITC) of three ELP fusion proteins were 2 M NaCl at 28 ℃. After two rounds of ITC, the three ELP fusion proteins were purified to more than 90% purities, which were comparable to that of affinity-purified PoIFNα-His and PoIFNγ-His. Cytopathic effect inhibition assay showed that the five rPoIFNs had potent but different antiviral activities against two different viruses on two different cell types. The plasma solubility assay showed that the three ELP-fused rPoIFNs remained as soluble proteins under the physical conditions. The plasma stability of three ELP-fused rPoIFNs was significantly improved in comparison with that of PoIFN-α. These data suggest that ELP fusion is a feasible strategy to enhance purification and plasma stability of rPoIFNs. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases affecting pig industry worldwide. Sialoadehesin (Sn) and CD163 are the two specific receptors for PRRSV infection of porcine alveolar macrophages. Our previous study showed that the soluble Sn receptor Sn4D-Fc and soluble CD163 receptor SRCR59-Fc expressed by the two recombinant adenoviral (rAd) vectors have an additive anti-PRRSV effect in vitro . In the present study, rAd-Sn4D-Fc and rAd-SRCR59-Fc were inoculated into pigs, and the efficient expression of Sn4D-Fc and SRCR59-Fc proteins was detected by ELISA. Then, PRRSV-naïve pigs were inoculated with rAd-Sn4D-Fc and/or rAd-SRCR59-Fc before contagious infection with different PRRSV strains. Among the three rAd inoculation groups, simultaneous inoculation with the two rAd vectors provided the best protection against highly pathogenic JXA1 strain PRRSV, followed by rAd-SRCR59-Fc inoculation and rAd-Sn4D-Fc inoculation. Clinical observation and quantitative RT-PCR analyses showed that all of the double rAd-inoculated pigs (n = 9) survived from the contagious infection with highly pathogenic JXA1, JS07 or SH1705 strain PRRSV with significantly alleviated clinical scores, viremia, fecal viral emission and tissue virus loads. These data suggest that rAd-Sn4D-Fc and rAd-SRCR59-Fc can be developed further as the universal therapeutic vaccine to facilitate PRRSV eradication. [ABSTRACT FROM AUTHOR]

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance. [ABSTRACT FROM AUTHOR]

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin, but its practical application is limited due to the complex purification procedure. In this study, the C2 domain of streptococcal protein G (SPG) with the binding affinity for chicken IgY was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein, and purified to a high purity by inverse transition cycling (ITC). Binding experiments showed that chicken IgY could bind to and eluted off the ELP-C2 fusion protein in pH-, temperature- and/or time-dependent manners. By using the ELP-C2 protein, a simple chicken IgY purification method was developed, and its purification performance was compared with that of ammonium sulfate precipitation and ethanol fractionation. Quantitative SDS-PAGE analysis showed that the ELP-C2 binding capture method provided a chicken IgY purity of 96.3% and a recovery of 64%, both of which were significantly higher than that of the two traditional methods The ELP-C2 binding capture method could be accomplished within 3 h, in contrast to 30.3 h for ammonium sulfate precipitation or 4.3 h for ethanol fractionation. These data suggest that the ELP-C2 binding capture was a simple, efficient and cost-effective method for purification of chicken IgY. [ABSTRACT FROM AUTHOR]

A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the 'spiked' PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples. [ABSTRACT FROM AUTHOR]

Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri , Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese dairy herd are different from the previously reported, and that the herd- or farm-based diagnosis of staphylococcal bovine mastitis is required. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease. As part of the development of RNA interference (RNAi) strategy against the disease, in this study a recombinant adenovirus (rAd) expressing the artificial microRNA (amiRNA) targeting the 3′ untranslated region (UTR) was used to investigate the exosome-mediated amiRNA transfer from different pig cell types to porcine alveolar macrophages (PAMs). Quantitative RT-PCR showed that the sequence-specific amiRNA was expressed in and secreted via exosomes from the rAd-transduced pig kidney cell line PK-15, PAM cell line 3D4/163, kidney fibroblast cells (PFCs) and endometrial endothelial cells (PEECs) with different secretion efficiencies. Fluorescent microscopy revealed that the dye-labeled amiRNA-containing exosomes of different cell origins were efficiently taken up by all of the five types of pig cells tested, including primary PAMs. Quantitative RT-PCR showed that the amiRNA-containing exosomes of different cell origins were taken up by primary PAMs in both time- and dose-dependent manners. Both quantitative RT-PCR and viral titration assays showed that the exosome-delivered amiRNA had potent anti-viral effects against three different PRRSV strains. These data suggest that the exosomes derived from pig cells could serve as an efficient miRNA transfer vehicle, and that the exosome-delivered amiRNA had potent anti-viral effects against different PRRSV strains. [ABSTRACT FROM AUTHOR]

Highlights: [•] We generated recombinant adenoviruses expressing soluble Sn or CD163 receptors as porcine Fc fusion proteins. [•] We demonstrated PRRSV binding activity for soluble receptors Sn4D-Fc and SRCR59-Fc. [•] We showed strong additive anti-PRRSV effect between the two soluble viral receptors. [•] We demonstrated efficient delivery of the soluble viral receptors to PAM via transduction of PRRSV non-permissive pig cells. [ABSTRACT FROM AUTHOR]

A robust artificial microRNA (amiRNA) strategy against porcine reproductive and respiratory syndrome virus (PRRSV) was developed by targeting the untranslated regions (UTRs). Six candidate amiRNAs targeting the 5′ or 3′ UTR were used for vector construction, and four effective amiRNAs were selected for further study using a vector transfection/virus infection assay. In cell cultures stably transfected with the four amiRNA vectors, expression of the sequence-specific amiRNAs was confirmed using poly(A)-tailed RT-PCR. After infection with three different PRRSV strains, the viral RNA genome and/or transcript were inhibited by ~90 % (semi-quantitative RT-PCR), and the viral titers were decreased by more than six log CCID (viral titration assay) before day 3 postinfection. The potent anti-PRRSV effects lasted for at least 5 days. Sequence analysis showed that the amiRNA antiviral activities were not compromised by the presence of one or two mismatches in their binding targets. This work constitutes a step towards developing a more effective RNAi strategy against PRRSV. [ABSTRACT FROM AUTHOR]

Abstract: RNA interference (RNAi) is a potent mechanism against a variety of viral infections. Infectious bursal disease virus (IBDV) causes an important disease economically in chickens, which is difficult to control. As part of the development of viral vector-mediated RNAi strategy against the disease, five anti-VP2 small interference RNAs were selected for construction of microRNA (miRNA) expression vectors tailored for avian cells. Transfection of DF-1 cells with the five vectors resulted in significant inhibition of VP2-EGFP reporter gene expression. More effective miVP2A and miVP2E were selected for further study using single or double miRNA expression vectors. After demonstration of specific miRNA expression, the gene silencing effects were determined in the vector-transfected and IBDV-infected cells. Reverse transcriptase PCR and virus titration showed inhibition rates from 76 to 82% on VP2 expression and significant decreases in virus titer by individual and co-expressed miVP2A and miVP2E. The inhibitory effects lasted for at least 120h after infection with IBDV. These data suggest that the miRNAs targeting the VP2 can inhibit efficiently replication of IBDV. [Copyright &y& Elsevier]

With the module of assimilating AMSU-A (Advanced Microwave Sounding Unit-A) and AIRS (Atmospheric Infrared Sounder) data in the WRFDA (Weather Research and Forecasting Model Data Assimilation) system, the impacts of joint assimilation of the radiance observations from two satellites on the simulation of typhoon Chan-hom (2015) are addressed. For comparison, experiments with the assimilation of solely GTS (Global Telecommunications System) data, AMSU-A data, or AIRS data are also performed. The results show that, compared to other experiments, the analysis field after assimilating multiple radiance data is closer to the observation. The simulated steering flow in its forecast field is conductive to the northeast twist of the typhoon. In addition, the simulated rainband and the FSS (fraction skill score) calculated from the experiment with assimilating multiple radiance data are better. In the deterministic forecast, better performance is obtained from the simulation with multiple radiance data in the forecast of track, MSLP (minimum sea level pressure), and MSW (maximum surface wind). [ABSTRACT FROM AUTHOR]

• We obtained PK-15 cell clones with different permissivity to PCV2 infection. • We constructed a transposon vector overexpressing pig interferon-gamma fused with IgG Fc domain. • We generated two PK-15 cell lines stably expressing IFNg-Fc fusion protein. • We demonstrated that the two cell lines can be used for PCV2 vaccine production. Porcine circovirus 2 (PCV2)-associated diseases affect the swine industry worldwide. Vaccination is the major tool for the disease control, but the vaccine production is hindered by lower propagation rate of PCV2 in vitro. Previous studies showed that interferons (IFNs) can increase PCV2 yield in PK-15 cells. In the present study, we constructed a Sleepy Beauty (SB) transposon vector expressing porcine IFNg gene fused with the coding sequence for immunoglobulin G Fc domain. After dilution cloning, the transposon and transposase vectors were co-transfected into PK-15 cell clones with higher permissivity to PCV2 infection. Two transgenic PK-15 cell lines, namely PK15-IFNgRan and PK15-IFNgSB which contained randomly integrated transfer vector or SB cassette without selection marker, were screened by PCR analysis. The characterization results demonstrated that the two transgenic cell lines can stably express IFNg-Fc fusion protein with potent antiviral activities. Both viral titration and quantitative PCR analyses showed that the two transgenic cell lines are highly permissive to PCV2 infection with significantly increased viral yields. These results indicate that the two transgenic PK-15 cell lines, PK15-IFNgSB in particular, can be used for PCV2 vaccine development. [ABSTRACT FROM AUTHOR]